PROJECT SUMMARY Significance: Genetic instability is involved in the etiology of a variety of diseases, and environmenal agents that damage DNA are contributing factors. UV irradiation and reactive oxygen species (ROS) produced in response to a variety of environmental agents contribute to genetic instability by generating mutagenic lesions that are preferentially distributed in the genome. In addition, genetically unstable endogenous DNA ?hotspots? often co-localize within or near DNA sequences that have the capacity to adopt alternatively structured DNA (i.e. non-B DNA) such as H-DNA. However, the mechansims involved in the generation of mutation hotspots are not clear. An objective of this project is to fill a critical gap in understanding why DNA damage and mutations occur within particular genomic hotspot regions. The results from the experiments outlined in this proposal will allow us to provide definitive evidence for the role of DNA structure-forming sequences in genetic instability and the generation of mutation hotspots in the presence of environmental DNA damaging agents. The results obtained will assist in achieving our long-term goal of reducing genetic instability caused by exposure to DNA damaging agents and the associated risks of disease. Hypothesis: We postulate that non-B DNA sequences are more susceptible to DNA damage by exogenous environmental factors, leading to increased mutagenesis. Preliminary Data: We have demonstrated that DNA damage caused by UV irradiation and ROS preferentially increases non-B DNA-induced mutagenesis in mammalian cells compared to B-DNA control sequences, implicating non-B DNA sequences in the generation of UV- and ROS-induced base damage and mutation hotspots. Furthermore, NextGen sequencing revealed H-DNA as a mutation hotspot in mammalian cells. Specific Aims: This project entails examining the impact of DNA base damage generated by environmental toxicants on genetic instability at endogenous mutation hotspots. In Aim 1, the impact of UV irradiation or ROS on non-B DNA-induced mutagenesis will be determined in mammalian cells across a range of damage levels and the mutation spectra will be characterized. In Aim 2, single-molecule, real-time (SMRT) sequencing will be used to detect and locate base lesions, with single-nucleotide resolution, induced by UV irradiation or ROS within canonical B-DNA versus mutagenic H-DNA sequences in vitro and in vivo. We will: (i) characterize the signature kinetic profiles of major DNA lesions produced by UV and ROS exposure; (ii) develop improved data analyses tools for lesion detection; and (iii) determine lesion location within non-B DNA versus B-DNA sequences following DNA damage. The results from these studies will allow us to determine the relationship between the accumulation of DNA base damage within structure-forming DNA sequences and the generation of mutation hotspots, which in turn will provide the foundation for the development of novel strategies to reduce genomic instability and related diseases.